High throughput sarcomeric assay

ABSTRACT

The present invention provides high throughput screening systems for identifying compounds that modulate the biological activity of a biochemically functional sarcomere. The method can be performed in plurality simultaneously with fluorescence or absorbance readouts.

FIELD OF THE INVENTION

The invention relates to methods for the identification of contractilemodulators of the cardiac sarcomere and use of such methods for theidentification of therapeutic agents.

BACKGROUND OF THE INVENTION

Congestive heart failure is a growing epidemic in our aging population.Its prevalence has been growing as the population ages and ascardiologists are more successful at reducing mortality from ischemicheart disease, the most common cause of congestive heart failure.Roughly 4.6 million people in the United States have heart failure withan incidence approaching 10 per 1000 after age 65 years. Hospitaldischarges for congestive heart failure rose from 377,000 in 1979 to957,000 in 1997 making congestive heart failure the most commondischarge diagnosis in people age 65 and over. The five year mortalityfrom congestive heart failure approaches 50%. Hospitalization for heartfailure is usually the result of inadequate outpatient therapy. Hence,while heart failure therapy has greatly improved over the last severalyears, new and better therapies are still required to improve thesestill dismal statistics.

Inotropes are drugs that increase the contractile ability of the heart.As a group, all current inotropes have failed to meet the gold standardfor heart failure therapy, that is, to prolong patient survival (FDACardiorenal Panel: Minutes Jan. 27, 1998 afternoon session,www.fda.gov). Despite this fact, intravenous inotropes continue to bewidely used in acute heart failure to allow for reinstitution of oralmedications or to bridge patients to heart transplantation, whereas inchronic heart failure, oral digoxin is used as an effective inotrope torelieve patient symptoms, improve the quality of life, and reducehospital admissions for heart failure.

Currently, there is a paucity of agents that can safely improve cardiacfunction; most agents have detrimental side effects if given for morethan a few days. As for chronic inotropic use, only digoxin has provensafe to administer even though it has a narrow therapeutic range. Themost recently approved short-term intravenous agent, milrinone, is nowover ten years old. The only available oral drug, digoxin, is over 200hundred years old. There is a great need for agents that exploit newmechanisms of action and may have better outcomes in terms of relief ofsymptoms, safety, and patient mortality, both short-term and long-term.The present invention provides methods for identifying such agents.

SUMMARY OF THE INVENTION

The present invention provides methods to identify candidate agents thatbind to a protein or act as a modulator of the binding characteristicsor biological activity of a protein. In one embodiment, the method isperformed in plurality simultaneously. For example, the method can beperformed at the same time on multiple assay mixtures in a multi-wellscreening plate. Furthermore, in a preferred embodiment, fluorescence orabsorbance readouts are utilized to determine activity. Thus, in oneaspect, the invention provides a high throughput screening system.

In one embodiment, the present invention provides a method ofidentifying a candidate agent as a modulator of the activity of a targetprotein complex. Preferably, the target protein complex either directlyor indirectly produces ADP or phosphate. More preferably, the targetprotein complex comprises a preparation comprising one or more of thefollowing proteins: myosin, actin, and cardiac regulatory proteins. In aparticularly preferred embodiment, the target protein complex is areconstituted sarcomere consisting of actin, myosin, and the cardiacregulatory proteins.

The method further comprises adding a candidate agent to a mixturecomprising the target protein complex under conditions that normallyallow the production of ADP or phosphate. The method further comprisessubjecting the mixture to an enzymatic reaction that uses said ADP orphosphate as a substrate under conditions that normally allow the ADP orphosphate to be utilized and determining the level of activity of theenzymatic reaction as a measure of the concentration of ADP orphosphate. The phrase “use ADP or phosphate” means that the ADP orphosphate are directly acted upon. In one case, the ADP, for example,can be hydrolyzed or can be phosphorylated. As another example, thephosphate can be added to another compound. As used herein, in each ofthese cases, ADP or phosphate is acting as a substrate. A change in thelevel between the presence and absence of the candidate agent indicatesa modulator of the target protein complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically demonstrates that the ATPase rate of thereconstituted system responds in a calcium dependent manner. Theconditions used are S1 at 1.25 μm, actin at 14 μm, Tn complex consistingof TnC, TnI, and TnT at 3 μm and Tm at 2 μm in a buffer containing 12 mMK-Pipes pH 6.8, 2 mM MgCl₂, 1 mM ATP, 1 mM DTT, and 0.1 mg/ml BSA. They-axis represents the rate of ATP hydrolysis.

FIG. 2. The top left panel and bottom left panel show the shift to theleft and upward of the calcium sensitivity curves for two compoundsassayed against a reconstituted sarcomere. The ATPase rate issignificantly increased at calcium levels greater than pCa=7. The curvesare normalized to the ATPase rate at pCa=4 in control. The top rightpanel demonstrates that Compound 1 activates cardiac but not skeletalmyosin. The bottom right panel shows the lack of effect of Compound 2when there are no regulatory complex proteins present suggesting thetarget of this compound is the regulatory complex.

FIG. 3. Diagrammatic representation of the isometric muscle fiberapparatus. Major individual components and their sources are: ForceTransducer, 400A Force Transducer (Aurora Scientific, Ontario, Canada);Data Acquisition, PCI-MIO-16E4 16 channel AID Board, 250 Khz, 12 bit(National Instruments, Dallas, Tex.); Software, Labview DevelopmentSystem National Instruments, Dallas, Tex.); Vibration Isolation, VHIsolation Workstation (Newport Corp, Irvine, Calif.); Microscope, ZeissStemi 2000-C Dissecting Microscope. Fiber Preparation is as follows: Pighearts are removed from freshly slaughtered pigs (˜1 hr) and transportedon ice. Trabeculae are dissected from the right ventricle, and placed inice cold High Relaxing (HR) solution containing 36 mM K-MOPS, 53.4 mMKCl, 7 mM MgCl2, 25 uM CaCl2, 10 mM EGTA, 10.6 MM creatine phosphate,5.4 mM ATP, 1 mM DTT, and 0.03 mg/ml creatine kinase (pH 7.0). Thinbundles of muscle fibers (1-2 mm in diameter) are dissected from thetrabeculae in HR solution, and the fibers are extracted overnight (12-16hours) at 4° C. in HR solution containing 1% Triton X-100 and a proteaseinhibitor cocktail. Fibers are then transferred to a 1:1 mixture of HRsolution and glycerol, also containing protease inhibitors, and storedat −20° C.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

“Target protein complex” refers to a protein or combination of proteinsthat directly or indirectly produces ADP or phosphate. A particularlypreferred target protein complex is a biochemically functional sarcomerepreparation comprising myosin, actin, tropomyosin, and the troponincomplex. As such, preferred target proteins include, but are not limitedto, cytoskeletal proteins including, but not limited to, myosins,actins, tropomyosins, and troponins. Suitable target proteins alsoinclude fragments of these proteins. In a preferred embodiment, one ormore of the target proteins is derived from mammalian cells.

The terms “isolated”, “purified”, or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

“Candidate agent” (used interchangeably herein with “test composition”and “test compound” and “test agent”) refers to a molecule orcomposition whose effect on the target proteins is desired to assay. The“candidate agent” can be any molecule or mixture of molecules,optionally in a suitable carrier.

By “ATPase” herein is meant an enzyme that hydrolyzes ATP. ATPasesinclude proteins comprising molecular motors such as myosins.

A “therapeutic” as used herein refers to a compound that is believed tobe capable of modulating the contractility of the cardiac sarcomere invivo that can have application in both human and animal disease.Modulation would be desirable in a number of conditions including, butnot limited to, congestive heart failure and diastolic heart failure.

II. The Target Protein Complex

-   -   A. Overview of the Sarcomere

Comprising nearly 60% of cardiac cell volume is the cardiac sarcomere.The sarcomere is an elegantly organized cellular structure found incardiac and skeletal muscle made up of interdigitating thin and thickfilaments. The thick filaments are composed of myosin, the proteinresponsible for transducing the chemical energy of ATP hydrolysis toforce and directed movement. Myosin and its functionally related cousinsare called motor proteins. The thin filaments are composed of a complexof proteins. Actin is a filamentous polymer and is the substrate uponwhich myosin pulls during force generation. Bound to actin are a set ofregulatory proteins, the troponin complex and tropomyosin, that make theactin-myosin interaction dependent on changes in intracellular Ca²⁺levels. With each heart beat, Ca²⁺ levels rise and fall, initiatingcardiac muscle contraction and then cardiac muscle relaxation (Robbins Jand Leinwand L A. (1999) Molecular Basis of Cardiovascular Disease,Chapter 8. editor Chien, K. R., W.B. Saunders, Philadelphia). Each ofthe components of the sarcomere contributes to its contractile response.

-   -   B. Myosin

The most extensively studied of all the motor proteins is myosin. Of thethirteen distinct classes of myosin in human cells, myosin-II is theform responsible for contraction of skeletal, cardiac, and smoothmuscle. This form of myosin is significantly different in amino acidcomposition and in overall structure from myosins in the other twelvedistinct classes (Goodson H V and Spudich J A. (1993) Proc. Natl. Acad.Sci. USA 90:659-663). Myosin-II consists of two globular head domains,called Subfragment-1 or S1, linked together by a long α-helicalcoiled-coiled tail. Proteolysis of myosin generates either S-1 or heavymeromyosin (HMM, a two-headed form with a truncated tail), depending onconditions. S1 contains the ATPase and actin-binding properties of themolecule. S1 has been shown to be sufficient to move actin filaments invitro (Toyoshima Y Y, Kron S J, McNally E M, Niebling K R, Toyoshima C,and Spudich J A. (1987) Nature 328:536-539), and is therefore clearlythe motor domain of the molecule.

The high resolution crystal structure for skeletal S1 is known in bothits putative pre-stroke and post-stroke states (Rayment I, Rypniewski WR, Schmidt-Base K, Smith R, Tomchick D R, Benning M M, Winkelmann D,Wesenberg G, Holden H M. (1993) Science 261:50-58 and Dominguez R,Freyzon Y, Trybus K M, Cohen C. (1998) Cell 94:559-571). S1 consists ofa globular actin-binding and nucleotide-binding region known as thecatalytic domain. This domain is attached at its carboxy-terminus to anα-helix that has two light chains of ˜20 kDa each wrapped around it.This light-chain binding domain of S1 is known as the lever arm. Upontransitioning from the pre-stroke to the post-stroke state of the S1,the lever arm swings through an angle of ˜90° about a fulcrum point inthe catalytic domain near the nucleotide-binding site. The “powerstroke” is driven by the hydrolysis of ATP.

The other end of the myosin molecule is an α-helical coiled-coiled tailinvolved in self assembly of myosin molecules into bipolar thickfilaments. These thick filaments interdigitate between thinner actinfilaments in the sarcomere, and the two filament systems slide past oneanother during contraction of the muscle. This filament slidingmechanism involves conformational changes in the myosin heads causingthem to walk along the thin actin filaments at the expense of ATPhydrolysis.

Mammalian heart muscle consists of two forms of cardiac myosin, α and β,and they are well characterized (Robbins, supra). The beta form is thepredominant form (>90 percent) in adult human cardiac muscle. Both havebeen observed to be regulated in human heart failure conditions at bothtranscriptional and translational levels (Miyata supra), with the alphaform being down-regulated in heart failure.

The sequences of all of the human skeletal, cardiac, and smooth musclemyosins have been determined. While the cardiac α and β myosins are verysimilar (93% identity), they are both considerably different from humansmooth muscle (42% identity) and more closely related to skeletalmyosins (80% identity).

Conveniently, cardiac muscle myosins are incredibly conserved acrossmammalian species. For example, both a and p cardiac myosins are >96%conserved between humans and rats, and the available 250-residuesequence of porcine cardiac β myosin is 100% conserved with thecorresponding human cardiac β myosin sequence.

-   -   C. The Regulatory Proteins

The regulatory proteins are a set of four proteins that bind to theactin filament and confer calcium regulation. More specifically, theyprevent myosin binding in the absence of calcium. Tropomyosin (Tm) is along coiled-coiled alpha-helix that sits in the groove of the actinfilament and sterically prevents myosin binding. The troponin complex iscomprised of three proteins, troponin C, troponin I, and troponin T(TnC, TnI, and TnT, respectively), that confer calcium sensitivity tothe actin filament.

TnC is the calcium sensor, containing four calcium binding sites. Thecrystal structure of its skeletal counterpart is solved (Houdusse A,Love M. L., Dominguez R, Grabarek Z, Cohen C. (1997) Structure5(12):1695-711). Two of the sites are exchangeable and become occupiedas intracellular calcium increases at the initiation of cardiaccontraction. Communicating via TnI and TnT, this calcium binding resultsin a movement of Tm out of the actin filament groove exposing bindingsites for the myosin motors (Lehman W, Vibert P, Uman P, Craig R. (1995)J Mol Biol 251(2):191-6). Muscle contraction ensues. At the conclusionin the cardiac cycle, the process reverses and relaxation ensues.

The troponins are only found in skeletal and cardiac muscle whiledifferent tropomyosin genes are expressed in many cell types. Thegenomics of tropomyosin and the troponins are complicated by the factthat there are many splice variants of tropomyosin and TnT. However,again the general rule is that conservation of cardiac sequences isextremely high across different species (90% identity or more) while inthe same species different tissues have more divergent sequences (<65%identity).

-   -   D. Actin

The complete sequences of the various actin isoforms have beenestablished, and they all show >98% identity (Vandekerckhove & Weber1979). Again, conservation across animal species is extremely high.Human cardiac a actin, for example, is 100% identical to its chickencounterpart. Unlike myosin though, human cardiac α actin only has fouramino acid changes when compared to human skeletal muscle actin.

A further embodiment of this invention provides for a method forproducing actin wherein actin is precipitated from a solution in thepresence of high concentration of a magnesium salt, preferably magnesiumchloride. Actin para-crystals which form under these conditions can beprecipitated at significantly lower centrifugation speed than those usedin conventional procedures for F-actin (for example, see J. D. Pardeeand J. A. Spudich, Purification of muscle actin, Methods in CellBiology, v. 24, pp.271-289, 1982). Additional advantage is thatpolymerization and wash are performed in a single step. Generally, aconcentration of at least 10 mM is used. More preferably, the magnesiumchloride will be present at a concentration of from about 25 to about100 mM; more preferably, from about 40 to about 60 mM. In a preferredembodiment, a concentration of 50 mM was used.

-   -   E. A Biochemically Functional-Sarcomere

In a particularly preferred embodiment, the target protein complex is abiochemically functional sarcomere preparation. The functionalbiochemical behavior of the sarcomere, including calcium sensitivity ofATPase hydrolysis, may be reconstituted from purified individualcomponents. Since all the regulatory components are present, this systemallows for simultaneous screening of the entire protein machinery atonce.

A particularly preferred target protein complex is a biochemicallyfunctional sarcomere comprising myosin, actin, tropomyosin, and thetroponin complex. As such, preferred target proteins include, but arenot limited to, cytoskeletal proteins including, but not limited to,myosins, actins, tropomyosins, and troponins. Suitable target proteinsalso include fragments of these proteins. In a preferred embodiment, thetarget proteins are from mammalian cells. More specifically, usingstandard purification techniques (Margossian S S and Lowey S. (1982)Methods Enzymol 85:55-71), adapted to yield larger quantities ofprotein, gram quantities of myosin, actin, and the regulatory proteinswere obtained. The myosin is treated with chymotrypsin to generate theS1 fragment; as opposed to intact myosin, S1 is a soluble protein atsalt concentrations necessary for ATPase determination. These proteinscan be combined in the proper ratios to reconstitute calcium regulatedmyosin ATPase activity with ATPase activation ratios of between about 5and 20 fold and more preferably of up to about 10 fold. This highlyregulated sarcomere can be prepared in reliably in large quantities andused for high throughput screening as described below.

III. High Throughput Screening

In a preferred embodiment, activity is measured by the methods disclosedin Ser. No. 09/314,464, filed May 18, 1999, which is incorporated hereinby reference in its entirety. These methods are preferably used inmultiwell plate formats and are ideally suited for high throughputscreening systems to identify lead compounds for medical therapeutic useand can also be used for diagnostics. More specifically, ADP orphosphate is used as the readout for protein activity.

The ADP or phosphate level can be monitored using coupling enzymesystems to result in changes in the absorbance or fluorescence of theassay mixture relative to a control mixture to determine if the testcompound or mixture of test compounds has an affect on the proteinfunction. This may be done with a single measurement but is preferablydone with multiple measurements of the same sample at different times.In the case of multiple measurements, the absolute rate of the proteinactivity can be determined, and such measurements have higherspecificity particularly in the presence of test compounds that havesimilar absorbance or fluorescence properties to that of the enzymaticreadout.

In a preferred embodiment, the target protein complex comprises abiochemically functional sarcomere. Compounds which modulate theactivity of the complex or one or more of the proteins thereof can befound by monitoring the production of either ADP or phosphate by avariety of methods.

There are several mechanisms by which candidate agents might modulatethe target protein complex. One might increase the rate at which myosinhydrolyzes ATP. Since, ATP hydrolysis is coupled to force production,this increase would be expected to increase the force of musclecontraction. In the presence of actin, myosin ATPase activity isstimulated >100 fold. Thus, ATP hydrolysis not only measures myosinenzymatic activity but also its interaction with the actin filament.Thus, a compound that modulates the function of the target proteincomplex is identified by an increase or decrease in the rate of ATPhydrolysis compared to a control assay in the absence of that compound.Preferred compounds will not activate force production or ATPaseactivity in the absence of calcium.

Alternatively, the force-pCa²⁺ relationship can be altered through theinteraction of the candidate agents with the regulatory complex.

In the case of the fully regulated sarcomere, the assay may be performedat any calcium concentration but preferably at calcium concentrationsbelow pCa=4. The calcium concentration chosen can prejudice the assaytowards discovering modulators of a specific protein in the complex. Forexample, at pCa=4 myosin modulators are more likely discovered as theregulatory proteins no longer exert much inhibitory influence over theATPase at the calcium concentration. Alternatively, in the presence ofEGTA (no calcium) compounds that activate the regulatory proteins in acalcium independent fashion may be found. These compounds may then beeliminated from further consideration as preferred compounds will notactivate force production or ATPase activity in the absence of calcium.Finally, actin and myosin may be screened in the absence of theregulatory proteins, thus only modulators of the actin-myosin systemwill be found.

One method for monitoring ADP production is to couple the ADP productionto NADH oxidation with the enzymes pyruvate kinase and lactatedehydrogenase and to monitor the NADH level either by absorbance orfluorescence (Nature 1956 178:632; Mol Pharmacol 1970January;6(1):31-40). One method for monitoring phosphate production isto use purine nucleoside phosphorylase to couple the phosphateproduction to the cleavage of a purine analog which results in either achange in absorbance (Proc Natl Acad Sci U S A 1992 Jun.1;89(11):4884-7) or fluorescence (Biochem J 1990 Mar. 1;266(2):611-4).With either method, the rate of ATP hydrolysis by the target proteincomplex of interest can be measured.

Test compounds can be assayed in a highly parallel fashion by usingmultiwell plates by placing the compounds either individually in wellsor testing them in mixtures. Assay components including the targetprotein complex, coupling enzymes and substrates, and ATP can then beadded to the wells and the absorbance or fluorescence of each well ofthe plate can be measured with a plate reader.

In a preferred embodiment, the method uses a 384 well plate format and a25 μL reaction volume. A pyruvate kinaseflactate dehydrogenase coupledenzyme system (Huang T G and Hackney D D. (1994) J Biol Chem269(23):16493-501, which is incorporated herein by reference) is used tomeasure the rate of ATP hydrolysis in each well. As will be appreciatedby those in the art, the assay components are added in buffers andreagents. Since the methods outlined herein allow kinetic measurements,the incubation periods are optimized to give adequate detection signalsover the background. The assay is done in real time giving the kineticsof ATP hydrolysis which increases the signal to noise ratio of theassay.

IV. Compounds Suitable for Screening

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic molecules having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. They include peptides,macromolecules, small molecules, chemical and/or biological mixtures,and fungal, bacterial, or algal extracts. Such compounds, or molecules,may be either biological, synthetic organic, or even inorganiccompounds, and may be obtained from several sources, includingpharmaceutical companies and specialty suppliers of libraries (e.g.,combinatorial libraries) of compounds.

Methods of the present invention are well suited for screening librariesof compounds in multiwell plates (e.g., 96-well plates), with adifferent test compound in each well. In particular, the methods may beemployed with combinatorial libraries. A variety of combinatoriallibraries of random-sequence oligonucleotides, polypeptides, orsynthetic oligomers have been proposed. A number of small-moleculelibraries have also been developed.

Combinatorial libraries may be formed by a variety of solution-phase orsolid-phase methods in which mixtures of different subunits are addedstepwise to growing oligomers or parent compounds, until a desiredcompound is synthesized. A library of increasing complexity can beformed in this manner, for example, by pooling multiple choices ofreagents with each additional subunit step.

The identity of library compounds with desired effects on the targetprotein complex can be determined by conventional means, such asiterative synthesis methods in which sublibraries containing knownresidues in one subunit position only are identified as containingactive compounds.

A screen of 20,000 compounds against the reconstituted sarcomere wasperformed at a pCa of 6.5, a calcium concentration reflective of what isnormally reached in viva during each cardiac cycle. Six compounds wereidentified that increased the sarcomere ATPase rate by 40% over controlor greater at a compound concentration of 10 μM. Three of the activatorswere myosin activators and were specific for cardiac myosin and did notappreciably activate skeletal myosin. The other three act on theregulatory apparatus as determined by assaying the compounds againstactin and myosin in the absence of the regulatory proteins. (FIG. 2).

While these compounds certainly alter the rate of ATP hydrolysis in abiochemical system, it was then determined whether these compounds wouldincrease the contractile force in a cardiac muscle fiber. The musclefiber is a densely packed highly ordered actin-myosin structure that islikely to act differently than a soluble biochemical system. Theseproperties were investigated by measuring contractile force in detergentpermeabilized cardiac fibers, also known as skinned cardiac fibers.These cardiac fibers retain their intrinsic sarcomeric organization butall aspects of cellular calcium cycling no longer exist. They have twoadvantages. First, the cellular membrane is not a barrier to compoundpenetration. Second, any increase in contractile force is related to thecompound's direct effect on the sarcomeric proteins rather than analteration in intracellular calcium levels since calcium concentrationis controlled. In addition, they have been used in the past toinvestigate the properties of this class of agents (Haikala H, NissinenE, Etemadzadeh E, Levijoki J, Linden I B. (1995) J Cardiovasc Pharmacol25(5):794-801 and Edes supra). Their disadvantage, however, is thatthese measurements are time consuming and have to be done carefully inan internally controlled manner since the fibers can be variable.

Tension measurements of muscle fibers are made by mounting the musclefiber at one end to a stationary post and mounting the other end to atransducer that can measure force (FIG. 3). After stretching the fiberto remove slack, the force transducer records increased tension as thefiber begins to contract. This measurement is called the isometrictension, since the fiber is not allowed to shorten. Activation of thepermeabilized muscle fiber is accomplished by placing it in a bufferedcalcium solution.

The contractile properties of the cardiac specific myosin activatorCompound 1 (FIG. 3) in skinned cardiac fibers have been determined. Thiscompound exhibited very little augmentation of force at low calcium orin the absence of calcium (the EGTA data point) and reasonable potency.

V. Additional Applications

In addition, the technology platform and secondary assays developedthrough this project would be useful for discovering agents to treat thelarge population with diastolic heart failure where the primarycontractile defect is in relaxation (Braunwald E. (1997) Heart Disease:A textbook of cardiovascular medicine. W.B. Saunders, PhiladelphiaChapters 12, 16, and 17). A second myosin motor, smooth muscle myosin,is also an attractive target protein complex. In this case, findingpotent modulators of the motor would find uses in treating conditionswhere smooth muscle contraction is part of the pathophysiologicalprocess. These disease areas include asthma, glaucoma, hypertension, andpre-term uterine labor.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety.

Experimental EXAMPLE 1

First, sufficient amounts of sarcomeric proteins from bovine heart werepurified. Standard methods of isolation were utilized and the finalproducts shown in FIG. 1. Myosin is purified by successive rounds ofsolubilization and precipitation in high and low salt buffers. Theconcentration was determined using an extinction coefficient ˜0.53cm²/mg. The myosin was then cut with chymotrypsin in the presence ofEDTA to generate the S1 fragment which is soluble at the low saltconditions optimal for ATPase activity (Margossian supra).

Actin was purified by first preparing an ether powder of cardiac muscle(Zot H G and Potter J D. (1981) Preparative Biochemistry 11:381-395) asdescribed below. Subsequently, actin was cycled between the filamentousand soluble state through rounds of centrifugation and dialysis (SpudichJ A and Watt S. (1971) J. Biol. Chem. 246:4866-4871). It was stored inthe filamentous state at 4° C.

Tropomyosin was extracted from the ether powder and separated from theother proteins based on pH dependent precipitations followed bysuccessive ammonium sulfate cuts at 53% and 65% (Smillie L B. (1981)Methods Enzymol 85 Pt B:234-41). The troponins were isolated as anintact complex of TnC, TnT, and TnI. Ether powder is extracted in a highsalt buffer. Successive ammonium sulfate cuts of 30% and 45% were done;the precipitate was solubilized by dialysis into a low salt buffer andthen further purified on a DEAE Toyopearl column with a 25-350 mM KClgradient. There was no measurable ATPase in any of the components exceptfor myosin which naturally had a very low basal ATPase in the absence ofactin.

Just prior to screening, the actin, tropomyosin and troponin complexwere mixed together in the proper ratio (7:1:1) to achieve maximalcalcium regulation of the actin filament (FIG. 1). The screen wasconducted at a pCa=6.5. This calcium concentration is in thephysiological range during muscle contraction and is above theinflection point on the pCa curve (FIG. 1). Thus, the steep slope of thecurve at this point will magnify the effect of ATPase activators thatshift calcium sensitivity to the left.

To measure the generation of ADP during the reaction, a pyruvatekinase/lactate dehydrogenase/NADH coupled enzyme system (PK/ILDH) wasadded to the actin. The myosin was kept separately. The plates are readin real time so that kinetic curves are obtained.

These compounds were in DMSO and were already spotted onto the bottomsof 384 well plates at 10 to 40 μg/ml final concentration.

Target class was then determined. Actin and myosin (in this case the S1fragment) were mixed with ATP, compound at 10 μM and the PK/LDH coupledenzyme system (FIG. 1). The ATPase rate in the presence of compound wascompared to control (an equivalent amount of DMSO). Hits that canactivate the myosin ATPase in the absence of the regulatory proteinsexert their effects against either actin or myosin (an example is shownin FIG. 1).

For myosin activators, selectivity for cardiac myosin versus skeletalmyosin was determined by substituting the cardiac myosin with skeletalmyosin. Rabbit cardiac and skeletal myosins were be used to avoid anyeffect of testing proteins from different species. Since the compoundswere identified against bovine myosin, this test also demonstrateswhether the compounds are isoform specific but not species specific.Finally, since cardiac actin was used in all assays, compounds thatactivate cardiac myosin but not skeletal myosin will formally beidentified as interacting with myosin rather than actin.

By elimination, hits that don't activate in the absence of theregulatory complex but do activate in the presence of the regulatorycomplex interact with this set of proteins to produce their effect.Specificity again will be tested by substituting homologous skeletalregulatory proteins for their cardiac counterparts. In the case of thetroponins, it will be possible eventually to demonstrate activityagainst the human targets as these are the only sarcomeric proteins thatcan be expressed easily in bacteria.

Each class of activators could have detrimental properties that can bedetermined by ATPase assay. The myosin hits could turn on the myosinATPase in the absence of actin, uncoupling the actin-myosin activatedATPase. This basal ATPase is easy to ascertain by measuring myosinATPase in the absence of actin. The regulatory hit compounds couldrelieve regulatory inhibition of myosin ATPase in the absence ofcalcium. ATPase assays of the regulatory activators in the presence ofEGTA will rule out calcium independent activation. These tests will beperformed as part of compound characterization.

All tension measurements are made using the following general protocol.Skinned fibers are further dissected from muscle bundles before use to˜0.3-0.6 mm diameter and approximately 0.5-1 cm in length. These fibersare mounted between the manual positioner and tension transducer usingcellulose acetate dissolved in acetone. Tension measurements are made at20-22° C., using buffers similar to HR solution, except for thesubstitution of 0.1 mM EGTA and various amounts of CaCl₂ to give pCavalues between 8 and 4. All solutions contain 5 mM K-phosphate (pH 7.0)to reduce maximal tension development and prolong fiber life andreproducibility. Fibers are stretched until tension begins to increasemeasurably, and then cycled several times between relaxing (0.1 mM EGTA)and pCa 4.0 to measure maximal tension development and ensure fiberintegrity. Baseline tension is subtracted from all measurements, and thesubsequent values are normalized to the maximal tension produced at pCa4.0 (100%). Measurements are made at various calcium concentrations(calculated using the program MAXChelator,http://www.stanford.edu/˜cpatton/maxc.html, that adjusts for thepresence of ATP and magnesium) in the presence and absence of compound.

EXAMPLE 2 Actin Preparation

-   -   1. Extract powder (as prepared in Example 3 or 4 below) with 20        ml buffer A (see below, add BME and ATP just prior to use in        each of the following steps) per gram of powder (200 ml per 10        g). Use a large 4L beaker for 150 g of powder. Mix vigorously to        dissolve powder. Stir at 4° C. for 30 min.    -   2. Separate extract from the hydrated powder by squeezing        through several layers of cheesecloth. Cheesecloth should be        pre-sterilized by microwaving damp for 1-2 min.    -   3. Re-extract the residue with the same volume of buffer A and        combine extracts.    -   4. Spin in JLA10 rotor(s) for 1 hr. at 10K rpm (4° C.). Collect        supernatant through 2 layers of cheesecloth.    -   5. Add ATP to 0.2 mM and MgCl₂ to 50 mM. Stir on stir plate at        4° C. for 60 minutes to allow actin to polymerize/form        para-crystals.    -   6. Slowly add solid KCl to 0.6 M (45 g/l). Stir at 4° C. for 30        min.    -   7. Spin in JLA10 rotor(s) at 10K rpm for 1 hr.    -   8. Depolymerization: Quickly rinse surface of pellets with        buffer A and dispose of wash. Soften the pellets by        pre-incubation on ice with small amount of buffer A in each tube        (use less than half of final resuspension volume total in all        tubes). Resuspend by hand first with cell scraper and combine        pellets. Wash tubes with extra buffer using a 25 ml pipette and        motorized pipettor, aggressively removing actin from sides of        tubes. Homogenize in large dounce in cold buffer A on ice. Use 3        ml per gram of powder originally extracted.    -   9. Dialyze against buffer A with 4 changes over 48 hour period.    -   10. Collect dialyzed actin and spin in the 45Ti rotor at 40 Krpm        for 1.5 hr. (4° C.).    -   11. Collect supernatant (G-Actin). Save a sample for gel        analysis and determination of protein concentration.

To polymerize G-actin for storage add KCl to 50 mM (from 3 M stock),MgCl₂ to 1 mM, and NaN₃ to 0.02% (from 10% stock). Store at 4° C. Do notfreeze.

Buffer A:

2 mM tris/HCl, 0.2 mM CaCl₂, 0.5 mM (36 ul/L) 2-mercaptoethanol, 0.2 mMNa₂ATP (added fresh), and 0.005% Na-azide; pH 8.0.

EXAMPLE 3 Powder Preparation

-   -   1. Volumes are given per ˜1000 g of the minced muscle.    -   2. Pre-cut and boil cheesecloth for 10 min. in water. Drain and        dry.    -   3. Mince chicken breast in a prechilled meat grinder.    -   4. Extract with stirring in 2 L of 0.1 M KCl, 0.15 M        K-phosphate, pH 6.5 for 10 min at 4° C. Spin 5000rpm, 10 min,        4° C. in JLA. Collect the pellet.    -   5. Extract pellets with stirring with 2 L of 0.05 M NaHCO₃ for 5        min. Spin 5000 rpm, 10 min, 4C in JLA. Collect the pellet.        Repeat the extraction once more.    -   6. Extract the filtered residue with 2 L of 1 mM EDTA, pH 7.0        for 10 min with stirring.    -   7. Extract with 2 L of H₂O for 5 min with stirring. Spin 10000        rpm, 15min, 4C in JLA. Carefully collect the pellet, part of        which will be loose and gelatinous.    -   8. Extract 5 times with acetone (2 L of acetone for 10 min each        with stirring). Squeeze through cheese-cloth gently. All acetone        extractions are performed at room temperature. Acetone should be        prechilled to 4° C.    -   9. Drying: Place the filtered residue spread on a cheese-cloth        in a large glass tray and leave in a hood overnight. When the        residue is dry, put in a wide mouth plastic bottle and store at        −20° C.

EXAMPLE 4 Alternate Powder Preparation

Based on Zot & Potter (1981) Prep. Biochem. 11(4) pp.381-395.

-   -   1. Dissect left ventricles of the cardiac muscle. Remove as much        of the pericardial tissue and fat as possible. Grind in a        prechilled meat grinder. Weigh.    -   2. Prepare 5 volumes of Extract buffer. Be sure the pH=8.0.        Then, homogenize the meat in a blender, 4×15 secs on blend with        15 secs in between. Do this with 1 volume (weight/volume) of        buffer taken from the 5 volumes already prepared. Add the        homogenate back to the extract buffer and stir until well mixed        (5 minutes).    -   3. Filter through one layer of cheese cloth in large        polypropylene strainer. Resuspend back into 5 volumes of extract        buffer as above.    -   4. Repeat step 3) 4 more times. At the end, do not resuspend in        extraction buffer but proceed to step 5). The pellets should be        yellow white.    -   5. Resuspend in 3 volumes (according to original weight) of 95%        cold Ethanol. Stir for 5 min and squeeze through cheesecloth as        above, repeat two more times.    -   6. Weigh squeezed residue and then resuspend in 3 volumes (new        weight/volume) of cold diethyl ether.    -   7. Repeat step 6) a total of 3 times.    -   8. Leave overnight in a single layer on a cheese cloth in a        glass tray.    -   9. When dry, collect the powder, weigh and store in a wide-mouth        jar at 4° C.

EXTRACT BUFFER: 50 mM KCl, 5 mM Tris pH 8.0

Prepare as 50× concentrate: For 2 L 250 mM Tris pH 8.0. Tris Base 121.14g/mol 60.6 g pH to 8.0 with conc. HCl, then add: 2.5 M KCl  74.55 g/mol 372 g

1-17. (canceled)
 18. A method of treating a condition characterized byabnormal smooth muscle contraction, the method comprising administeringa compound that modulates smooth muscle myosin contractility to apatient having or susceptible to the condition, wherein the compound ischaracterized by: a) adding a test compound to an in vitro mixturecomprising a target protein complex that directly or indirectly producesADP or phosphate under conditions which normally allow the production ofADP or phosphate; b) subjecting the mixture to an enzymatic reactionthat uses the ADP or phosphate as a substrate under conditions whichnormally allow the ADP or phosphate to be utilized; and c) determiningthe level of activity of the enzymatic reaction, wherein a change in thelevel between the presence and absence of the test compound indicatesthat the test compound is suitable for use as the compound in treating acondition characterized by abnormal smooth muscle contraction.
 19. Themethod of claim 18, wherein said compound increases myosin ATPase rateby at least 40% over control at an in vitro compound concentration of 10μM.
 20. The method of claim 18, wherein said determining comprisesdetecting fluorescence, luminescence, radioactivity, or absorbanceassociated with the enzymatic reaction.
 21. The method of claim 18,wherein said level of activity of said enzymatic reaction is determinedat multiple time points.
 22. The method of claim 18, wherein a pluralityof test compounds are added to the in vitro mixture.
 23. The method ofclaim 18, wherein said target protein complex directly producesphosphate or ADP.
 24. The method of claim 18, wherein the enzymaticreaction occurs at a calcium concentration below pCa equal to
 4. 25. Themethod of claim 18, wherein the enzymatic reaction occurs at a calciumconcentration of about pCa equal to
 4. 26. The method of claim 18,wherein the enzymatic reaction occurs at a calcium concentration ofabout pCa equal to 6.5.
 27. The method of claim 18, wherein theenzymatic reaction occurs in the presence ofethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA).
 28. The methodof claim 18, wherein said compound does not activate skeletal myosin.29. The method of claim 18, wherein the condition characterized byabnormal smooth muscle contraction is selected from asthma, glaucoma,hypertension, and pre-term uterine labor.